This invention relates to the production of polyclonal and monoclonal antibodies to specific sites of cyclosporine and/or cyclosporine metabolites, derivatives and analogues. The reactivity of these polyclonal and monoclonal antibodies makes them particularly useful for immunoassays for therapeutic drug monitoring (TDM). These immunoassays or TDM kits may include polyclonal or monoclonal antibodies to specific sites of cyclosporine (CSA) and/or metabolites, derivatives and analogues of cyclosporine. These kits may also include various combinations of polyclonal antibodies, polyclonal and monoclonal antibodies or a panel of monoclonal antibodies.
Cyclosporine is an 11-amino acid cyclic peptide of fungal origin (isolated from the fungus Tolypocladium inflatum) that contains two uncommon amino acids: (4R)-4-((E)-2butenyl)-4, N-dimethyl-1-threonine (Bmt) and 1-alpha-aminobutyric acid (Abu), as well as several peptide bond N-methylated residues (residues 1, 3, 4, 6, 9, 10, and 11). The structure of cyclosporine is given in FIG. 1.
Currently, the two immunosuppressive drugs administered most often to prevent organ rejection in transplant patients are cyclosporine (CSA) and tacrolimus (FK-506 or FK). Rapamycin (Rapa) is another known immunnosuppressant. Cyclosporine""s primary target appears to be the helper T lymphocytes. Cyclosporine acts early in the process of T cell activation, it has secondary effects on other cell types that are normally activated by factors produced by the T cells. Cyclosporine inhibits the production of interleukin 2 (IL-2) by helper T cells, thereby blocking T cell activation and proliferation (amplification of immune response). It is effective both in the prevention and in the treatment of ongoing acute rejection. The current model for the mechanism of action of CSA suggests that, in the T cell cytoplasm, CSA binds to a specific binding protein called immunophilin. The CSA-immunophilin complex in turn binds to and blocks a phosphatase called calcineurin. The latter is required for the translocation of an activation factor (NF-ATc) from the cytosol to the nucleus, where it would normally bind to and activate enhancers/promoters of certain genes.
In the presence of CSA, the cytosolic activation factor is unable to reach the nucleus, and the transcription of IL-2 (and other early activation factors) is strongly inhibited. As a result of this inhibition, T cells do not proliferate, secretion of gamma-interferon is inhibited, no MHC class II antigens are induced, and no further activation of the macrophages occurs.
Various side effects are associated with cyclosporine therapy, including nephrotoxicity, hypertension, hyperkalemia, hypomagnesemia and hyperuricemia. Neuro- or nephrotoxicity has been correlated with certain cyclosporine metabolites. A necessary requirement of cyclosporine drug monitoring assays is to measure the levels of parent cyclosporine drug and metabolite with immunosuppressive and toxic activity. There is a need for improved methods of monitoring levels of CSA and/or CSA metabolites and derivatives.
The current invention is drawn to methods for the preparation of immunogenic conjugates which elicit antibodies with specificity for cyclosporine related compounds. For the purposes of this application, the term cyclosporine related compound is meant to include any or all of the cyclosporine molecule itself and/or various cyclosporine metabolites and derivatives. Cyclosporine and cyclosporine metabolite conjugate immunogens are prepared and used for the immunization of a host animal to produce antibodies directed against specific regions of the cyclosporine or metabolite molecules. By determining the specific binding region of a particular antibody, immunoassays which are capable of distinguishing between the parent molecule, active metabolites, inactive metabolites and other cyclosporine derivatives/analogues are developed. The use of divinyl sulfone (DVS) as the linker arm molecule for forming cyclosporine/metabolite-protein conjugate immunogen is described.
In a first aspect, the invention provides antibodies which are capable of binding to a cyclosporine related compound. Such antibodies which recognize a specific region of said cyclosporine related compound or the CSA metabolites AM1 or AM9 are preferred. Monoclonal antibodies are most preferred. Also provided are methods for producing an antibody which is capable of recognizing a specific region of a cyclosporine related compound, said methods comprising: a) administering an immunogen comprising a cyclosporine related compound, a linker arm molecule and a protein carrier to an animal so as to effect a specific immunogenic response to the cyclosporine related compound; b) recovering an antibody to said cyclosporine related compound from said animal; and c) identifying the antibody binding region by comparing the reactivity of the antibody to a first cyclosporine related compound to the reactivity of the antibody to a second cyclosporine related compound. Such methods wherein said linker arm molecule is divinyl sulfone and where the cyclosporine related compound is linked to the carrier at amino acid residue 1 or 9 are preferred. The protein carrier may preferably be keyhole limpet hemocyanin or human serum albumin. Use of hybridoma cells to accomplish the above methods is also provided.
In another aspect, the invention provides immunoassay methods for measuring the level of a cyclosporine related compound in a mammal, comprising: a) incubating a biological sample from said mammal with an antibody which is capable of binding to a cyclosporine related compound; and b) measuring the binding of cyclosporine related compound to said antibody. Use of antibodies which recognize a specific region of said cyclosporine related compound or the CSA metabolites AM1 or AM9 in these assays is preferred. Use of monoclonal antibodies is most preferred. Immunoassay kits for measuring the level of a cyclosporine related compound in a biological sample, said kits comprising an antibody as described above are also provided. Also provided are assay methods for determining the amount of a particular cyclosporine related compound in a sample, comprising: a) contacting said sample with a first antibody according to claim 1; b) contacting said sample with a second antibody according to claim 1; and c) determining the amount of said particular cyclosporine related compound bound to said second antibody.